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수의학석사 학위논문

Heavy metal concentration and food

safety of shark meat in Jeju Island,

Republic of Korea

제주도 연근해 상어육의 중금속

농도 및 식품안전성

2019년 8월

서울대학교 대학원

수의학과 수의병인생물학 및 예방수의학 전공

김 상 화

Heavy metal concentration and food

safety of shark meat in Jeju Island,

Republic of Korea

By

Sang Wha Kim

August, 2019

Veterinary Pathobiology and Preventive Medicine

Department of Veterinary Medicine

The Graduate School of Seoul National University

수의학석사 학위논문

Heavy metal concentration and food safety of shark meat in Jeju Island, Republic of Korea

제주도 연근해 상어육의 중금속

농도 및 식품안전성

지도교수: 박 세 창

이 논문을 수의학 석사 학위논문으로 제출함

2019년 4월

서울대학교 대학원

수의학과 수의병인생물학 및 예방수의학 전공

김 상 화

Heavy metal concentration and food safety of shark meat in Jeju island,

Republic of Korea

By

Sang Wha Kim

Supervisor: Prof. Se Chang Park, D.V.M., Ph.D.

A dissertation submitted to the faculty of the Graduate School

of Seoul National University in partial fulfillment of the

requirements for the degree of Master in Veterinary

Pathobiology and Preventive Medicine

August, 2019

Veterinary Pathobiology and Preventive Medicine

Department of Veterinary Medicine

The Graduate School of Seoul National University

i

Heavy metal concentration and food

safety of shark meat in Jeju island,

Republic of Korea

Sang Wha Kim

(Supervised by Professor Se Chang Park)

Department of Veterinary Pathobiology and Preventive Medicine

College of Veterinary Medicine

The Graduate School of Seoul National University

Abstract

Shark meat is consumed as a food source worldwide, especially in Asian

countries. However, since sharks are apex predators in the ocean food chain, they

are prone to bioaccumulation of heavy metals. More than 100 million sharks are

caught annually for human consumption, and the safety of shark meat cannot be

overemphasized. Here, we examined heavy metal concentration in the muscle

tissue of 6 shark species including 3 migratory species (Carcharhinus brachyurus,

Carcharhinus obscurus, and Isurus oxyrinchus) and 3 local species (Triakis

ii

scyllium, Mustelus manazo, and Cephaloscyllium umbratile) from fish markets in

Jeju Island, Republic of Korea. The concentrations of 11 heavy metals (Cr, Fe, Cu,

Zn, As, Se, Cd, Sn, Sb, Pb, and Hg) and MeHg were analyzed. The result showed

that the average concentrations of all metals, except for that of As, were below the

regulatory maximum limits of many organizations, including the Codex standard.

Hg and MeHg were significantly correlated with body length, body weight, and

age, and the concentration of Hg was expected to exceed the limit in C.

brachyurus with a body length or weight of over 130 cm or 25 kg, respectively.

Our results indicate that shark meat can expose consumers to a high level of As

and that copper sharks bigger than the predicted size should be avoided for

excessive Hg. Considering these findings, a detailed guideline on consumption of

meat of different shark species should be suggested based on further investigation.

Keywords: Shark meat, Heavy metal, Food safety, Bioaccumulation,

Carcharhinus brachyurus

Student number: 2016-26230

iii

CONTENTS

Abstract∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙ i

Contents∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙ iii

Abbreviations∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙ iv

Introduction∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙ 1

Material & Methods∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙ 7

Results∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙ 16

Discussion∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙ 26

References∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙ 47

Summary∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙ 55

Abstract in Korean ∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙ 56

List of articles∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙ 58

List of conferences∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙ 62

Acknowledgements∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙ 66

iv

Abbreviations

Cr Chromium

Fe Iron

Cu Copper

Zn Zinc

As Arsenic

Se Selenium

Cd Cadmium

Sn Tin

Sb Antimony

Pb Lead

Hg Mercury

MeHg Methylmercury

IUCN International Union for Conservation of Nature

TBL Total body length

BW Body weight

ICP-MS Inductively coupled plasma mass spectrometry

CRM Certified reference material

DL Detection limit

DMA Direct mercury analyzer

GC Gas chromatography

CVAFS Cold vapor atomic fluorescence spectroscopy

MANOVA Multivariate analysis of variance

MVN Multivariate normality

v

MVR Multivariate regression

DOH Department of Health

SCF Scientific Committee on Food

FAO Food and Agriculture Organization of United States

JECFA Joint FAO/WHO Expert Committee on Food Additives

EU European Union

KFDA Korea Food & Drug Administration

UNEP United Nations Environment Programme

1

Introduction

Shark meat has been used as a food source since the fourth century, and

there was a drastic increase in its usage after World War I [1]. World shark capture

production and shark meat trade amounts have increased continuously since 1950s,

reaching the highest values on records in 2011 [2, 3]. Shark population thus has

decreased due to unregulated shark fishing worldwide [4]. Currently, 28% of non-

data deficient shark species are endangered according to the International Union

for the Conservation of Nature (IUCN) red list [5]. Although the capture amount

has decreased since 2011, the reason is still unclear whether there has been a

decrease in demand or a decrease in population [6]. Asia is a major contributor to

the global chondrichthyan market. Indeed, Japan, China, Taiwan, Hong Kong,

Singapore, Republic of Korea, Thailand, India, Indonesia, and Malaysia are all

involved in the production, import, and export of shark meat [1]. Asian countries

accounted for 59% of the global shark trade from 1976 to 2015; this percentage

has increased to 63% in the last 15 years [3].

2

In the Republic of Korea, ancient records on shark products as food

sources or clinical medicines include Shinjeung-donggukyeojiseungram

(新增東國輿地勝覽, Augmented Geographical Survey of Korea, 1481),

Donguibogam (東醫寶鑑, Principles and Practices of Eastern Medicine, 1613),

Jangjeon Ganchal (張瑱 簡札, Jangjeon’s letter, 1690), and Jasaneobo (玆山魚譜,

Record of Fish in Heuksan Island, 1814), and shark meat continues to be a major

food source at present. The amount of shark meat imported into Korea surged in

the 1990s and has remained steady at over 20,000 tons since 2003. Moreover, the

import value has increased, even after 2003, and was reported to be 85 million

USD in 2015 [3]. This increase in demand can be explained by the routine intake

of shark meat in Korea, both as a traditional dish in Gyeongsangbuk-do and as a

general cuisine in the form of steamed meat, roasted meat, soup, and skewers in

various provinces.

Fish meat consumption has various advantages, including reducing the

risk of cardiovascular disease and osteoporosis and facilitating the development of

embryos via increased intake of long-chain n-3 fatty acids [7]. However, various

3

environmental toxins including dioxin, polychlorinated biphenyls, and heavy

metals can also be introduced into the body by consuming fish meat. Toxins can

accumulate in the fish body via pollution sources such as industrial waste waters

from mine and refinery [8-10]. Among various toxins, heavy metal accumulation

is a critical parameter for establishing food safety of shark meat because of the

multiple harmful effects of heavy metals. Fish meat is the main source of mercury

(Hg), arsenic (As), copper (Cu), selenium (Se), lead (Pb), and cadmium (Cd) in

diet. Many national and international organizations have developed regulatory

maximum limits for different heavy metals to avoid damage to the body by fish

consumption [11].

Shark is a representative apex predator of marine ecosystems and its meat

has a higher risk of bioaccumulation of environmental toxins than other fish

species [12]. Since shark meat has been used as an important food source for a

long time, a large number of studies have been conducted on heavy metal

concentration to ensure food safety [8, 9, 12-21]. The concentrations of various

heavy metals have been measured at the level of species, habitat, environmental

4

conditions, and biological conditions (age, body length, body weight, etc.). Based

on the accumulated information, the food safety of shark meat is being assessed.

Among various heavy metal species, methylmercury (MeHg) especially

shows a good absorption rate (90%) and long retention time (half-life: 70 days) in

the human body. Excessive accumulation of MeHg can induce atrophy of the

cerebral cortex, ataxia, hearing loss, decreased visual acuity, and increased

incidence of cardiovascular diseases. The main source of MeHg in humans is fish

meat intake because MeHg is biomagnified through the marine and freshwater

food chain. Indeed, terrestrial animals typically have about 20 μg/kg MeHg,

whereas large fish species, such as tuna or sharks, can have MeHg concentrations

of up to 1 mg/kg [7, 22]. MeHg concentrations in fish depend on many factors,

including the concentrations of water and sediments, the pH and redox potential

of water, and the age, species, and size of fish [23]. Therefore, further studies are

necessary to ensure the safety of fish meat consumption, especially meat from

apex predators.

Carcharhinus brachyurus (copper shark), making up 60% of the shark

5

species used in this study, are pelagic, oceanic, and highly migratory species that

are distributed all over the coastal areas of tropical and temperate seas. They have

been fished commercially in many countries worldwide including New Zealand,

Australia, South Africa, Brazil, Uruguay, Argentina, Mexico, China, and Korea

[24, 25]. They migrate to the north in the spring and summer and migrate back to

the south in the fall and winter, reaching the sea around Jeju island from October

to February. These sharks consume cephalopods, pelagic and benthic teleosts, and

small elasmobranchs as their main food, staying at the top of the ocean food chain

and are by-caught in the Jeju island mainly with Japanese amberjack (Seriola

quinqueradiata), longtooth grouper (Epinephelus bruneus), and largehead hairtail

(Trichiurus lepturus) [25, 26]. They are sold at wholesale fish markets,

transported to other areas, and consumed by local people. Since they are being

caught and consumed not only in Korea but also in all countries, the food safety of

copper shark meat should be a concern. As they are the apex predators of the

ocean food chain, the need for bioaccumulation analysis is particularly

emphasized.

6

Accordingly, in this study, we evaluated the concentrations of heavy

metals (Cr, Fe, Cu, Zn, As, Se, Cd, Sn, Sb, Pb, Hg, and MeHg) in six shark

species including C. brachyurus captured at Jeju island (C. brachyurus,

Carcharhinus obscurus, Isurus oxyrinchus, Triakis scyllium, Mustelus manazo,

and Cephaloscyllium umbratile). We also examined correlations between metal

concentrations and biological criteria including age, sex, total body length (TBL),

body weight (BW), and habitat in C. brachyurus. With these results, we evaluated

the safety of consuming the meat of these six shark species.

7

Materials & Methods

Sampling

Sharks were sampled at Moseulpo and Hallim fish markets in Jeju, Korea

from October 2017 to December 2017, immediately after being released to the

markets from fishing ships, targeting the ‘shark meats actually being sold at the

fish markets’ as our sample population (Fig 1). For the Moseulpo fish market, a

total of 19 sharks were by-caught from the sea area between Gapa island and

Mara island by line fishing ships for Japanese amberjack (Seriola quinqueradiata),

angling fishing ships for largehead hairtail (Trichiurus lepturus), and longline

fishing ships for longtooth grouper (Epinephelus bruneus). All these 19 sharks

were sampled immediately after being auctioned. For the Hallim fish market,

sharks were by-caught by drift gillnets, and six of these sharks were sampled.

Almost every shark brought to the markets during the sampling period was

sampled to obtain the shark meat population actually consumed by the local

people. All samples were numbered from SNU-MO-0001 to SNU-MO-0025 in

8

chronological order. The species of the sharks were determined with their features

based on previous studies [27, 28]. Sharks were sampled and necropsied to

determine their health statuses within 12 h of being caught. TBL, BW, and the

girth right in front of the first dorsal fin were measured. The epaxial muscle and

4–6 vertebrae were sampled at the level of the first dorsal fin. Muscles were

sampled using a Whirl-Pak (Sigma-Aldrich, Germany) and kept frozen at -20 ℃

until analysis. Muscle tissues on the vertebrae were roughly removed, and all

vertebral joints were disparted. The neural arch and transverse processes were

removed from the centrums. Remnant tissue was removed after the centrums were

immersed in commercial sodium hydroxide solution. Then the centrums were

rinsed with water and dried for more than 72 h.

Age determination

Completely dried centrums were embedded with EPOXY Resin (Pace

Technologies, AZ, USA) and EPOXY Hardener (Pace Technologies). After 24 h

of hardening, centrums were sectioned using an Isomet low-speed saw (Buehler,

9

IL, USA) with a diamond saw into 2-mm-thick pieces. Samples were then

attached to slide glasses using 7036.20 Blanchard Wax (J.H. Young Company, NY,

USA), and then the centrum pieces were ground using an AutoMet 250 grinder-

polisher (Buehler) with sandpaper until the slices were thinner than 200 µm.

Samples were ground in two stages using different grinding papers (Carbimet 320

grit and 600 grit) to minimize the surface scratch. The annulus on the corpus

calcareum was measured on the slides independently by three researchers.

Heavy metal concentration analysis

Total concentrations of 10 heavy metals (chromium [Cr], iron [Fe],

copper [Cu], zinc [Zn], arsenic [As], selenium [Se], cadmium [Cd], tin [Sn],

antimony [Sb], and lead [Pb]) were analyzed using inductively coupled plasma

mass spectrometry (ICP-MS). Approximately 0.1 g of homogenized shark muscle

in wet condition was completely disintegrated by adding 10 mL HNO3 and

heating at 200 for 2 h. Samples were cooled to room temperature, deionized ℃

water was added to the digested samples, and the dilution factors were calculated

10

by measuring the final weight. ICP-MS was conducted with an Agilent 770x ICP-

MS (Agilent Technologies, CA, USA). Analysis was carried out in He mode with

a low matrix concentration, and the CeO/Ce ratio was 0.9%. Certified reference

materials (CRMs; VHG Labs, NH, USA) were used at a concentration of 0.1–100

ppb for calibration (Pearson’s r > 0.999). Analysis results were presented in mg/kg,

and the detection limit (DL) was 0.0001 mg/kg (KOPTRI, Seoul, Korea).

For total mercury analysis, shark muscle tissues were cut into 1-cm3

pieces and freeze-dried for 72 h using a freeze-dryer (FDU-1200; Eyela Co.,

Japan). The moisture composition ratio was calculated using the mass difference

between before and after freeze-drying. Freeze-dried samples were homogenized

thoroughly using a mortar, placed into conical tubes, and sealed with parafilm

until use. For total mercury analysis, approximately 0.01 g of freeze-dried and

homogenized shark muscle samples was weighed on a precision scale connected

to a Milestone DMA-80 Direct Mercury Analyzer (Milestone, Bergamo, Italy) and

analyzed. DOLT-5 (National Research Council Canada, Ottawa Ontario, Canada)

was used as a CRM, and the average recovery of CRM was 103.7% (± 9.8%, n =

11

13). The DL measured by DMA-80 was 0.06 ng/g. Dry weight concentrations

were converted into wet weight (WW) concentrations using the moisture

composition ratio.

For methylmercury analysis, approximately 0.01 g freeze-dried and

homogenized shark muscle sample and DOLT-5 CRM were weighed on a

precision scale and placed in glass bottles that had been washed with 15% HCl

(12 N) solution. After adding 5 mL of 25% KOH solution diluted with methanol,

samples were incubated in an oven at 60 over 12 h. Samples were cooled to ℃

room temperature, and deionized water was added to the sample solution for

dilution. Next, 200 mL of deionized water was added to the bubbler, and 1 mL

acetate buffer (2.0 M) was added to adjust the pH to 4.9. Diluted sample solution

and 0.1 mL of 1% tetraethylborate solution were added to the bubbler to produce

volatile methylethylmercury. Volatile methylethylmercury was collected into a

Tenax trap after 15 min of N2 gas purging and 15 min of incubation for ethylation

to methylated Hg species. The traps were then dried with N2 gas for 9 min. Hg

species were released from the Tenax trap following 30 s of heating, separated

12

into individual species using gas chromatography (GC), and detected by cold

vapor atomic fluorescence spectroscopy (CVAFS) using a Brooks Rand Model III

detector (Brooks Rand Instruments, WA, USA). The average recovery rate of

CRM was 101.6% (± 8.1%, n = 9). Dry weight concentrations were converted to

WW concentrations using the moisture composition ratio.

Statistical analysis

When non-detects that have a lower concentration than that of DL

comprise less than 15% of the data, replacement with 1/2DL is suggested as one

of the satisfactory methods for further analysis [29, 30], and based on this

suggestion, many papers have used the 1/2DL substitution method for statistical

analysis of data with non-detects [31-33]. Therefore, for a more detailed statistical

analysis other than simple average comparison, 1/2DL was used as a substitute for

non-detects, and only the heavy metals in which the non-detect portion was lower

than 15% were analyzed (Fe, Cu, Zn, As, Se, Hg, and MeHg). In cases of

biological data acquisition failure due to field situations, the related data were

13

excluded from statistical processing.

Multivariate analysis of variance (MANOVA) was used to determine

whether species, sex, and habitat affect heavy metal concentrations. Since

MANOVA analysis assumes both multivariate normality and homogeneity of

variance-covariance matrix of residuals and the raw data did not satisfy the

assumptions (Table 1), multivariate Box-Cox transformation was performed using

estimated transformation parameters (Table 2) [34]. Since the transformation was

applied to practical situations, estimated power parameters were replaced by

simple power parameters, i.e., 0, ±0.25, and ±0.5. The Box-Cox family requires

the responses to be strictly positive. Transformation was performed using the

powerTransform function in Car version 3.0-2 package in R [35]. is the power

parameter in the following equation.

Shapiro-Wilk test for marginally normality test, Bartlett’s test for

homogeneity of variance-covariance matrix assumption check, Mardia’s

14

multivariate normality (MVN) test, and Henze-Zirkler’s MVN test for jointly

normality tests were performed using statistical package R [35]. MVN tests were

performed with MVN package version 5.5 in R [36]. Normal Q-Q plots were also

generated to check normality. Since the assumptions were satisfactory after proper

transformation (Table 3, 4), MANOVA was conducted with car package version

3.0-2 in R. Data of C. umbratile was excluded when MANOVA was performed

for species due to the small sample number (sample number was only one).

Multivariate regression (MVR) was also performed to confirm whether

there is any significant correlation between age, BW, girth, TBL and each heavy

metal concentration using the lm function in R. To satisfy the fundamental

assumptions of multivariate linear regression, the raw data were transformed

using multivariate Box-Cox transformation for each covariate: age, BW, TBL, and

girth. All responsible variables were tested with Shapiro-Wilk test, Bartlett’s test,

Mardia’s MVN test, and Henze-Zirkler’s MVN test, and we confirmed that they

satisfy the assumptions.

To elucidate the correlations among parameters, excluding species

15

differences, MANOVA and MVR were conducted using data from C. brachyurus

only. Pearson’s coefficients and p values were also calculated using R to confirm

statistical significance [35]. Based on the results from MVR showing significant

correlation between heavy metal concentration and biological parameters of C.

brachyurus, linear and polynomial regressions were used and compared to each

other to identify a more, well-fitting model using OriginPro 8.5 (OriginLab

Corporation, Northampton, USA).

16

Results

Characteristics of the sampled sharks

In total, 25 sharks of six species were collected over a 3-month period. C.

brachyurus (copper shark), C. obscurus (dusky shark), and I. oxyrinchus (shortfin

mako) were by-caught by line fishing, angling fishing, and longline fishing in the

Moseulpo sea, and T. scyllium (banded houndshark), M. manazo (starspotted

smooth-hound), and C. umbratile (blotchy swell shark) were by-caught by drift

gillnets in the Hallim sea area. We could not collect some of the biological data

due to circumstantial causes. Collected biological data are summarized in Table 5

by species, and all the raw data are recorded in the Table 6. Because sharks longer

than 2 m are difficult to draw back to land when by-caught, sharks distributed in

fish markets are generally less than 2 m in length. Indeed, the sharks collected for

this investigation were all shorter than 2 m. Thus, for shark species that often have

mature lengths of over 2 m, including C. brachyurus, C. obscurus, and I.

oxyrinchus, only juvenile sharks were collected. As a result of age estimation

17

using vertebral section, all sharks were found to be younger than 7 years old

(Table 5). Based on this information, the sampled batch was not a true

representation of C. brachyurus, C. obscurus, and I. oxyrinchus. However, since

the collected specimens were subsamples of sharks that are actually consumed by

the local people, this batch was a good representation of shark meat as a food

material, which is the target sample population of this research. Moreover, since

these three species (C. brachyurus, C. obscurus, and I. oxyrinchus) show seasonal

migration and can be consumed in other countries on their migration route, the

importance of their food safety becomes greater.

Average heavy metal concentrations

The average moisture composition ratio was 74.6%, and the dry weight

concentrations were converted to WW concentration using this value. The average

concentrations of the 11 metals and MeHg in all 25 sharks are indicated in Table 7

and showed the following decreasing order: Fe > As > Zn > Cu > Se > Hg >

MeHg > Cr > Pb > Sn > Sb > Cd. The average concentrations of Cr, Sn, and Pb

18

were the highest in C. obscurus, whereas the average concentrations of Fe, Cu,

and Hg were the highest in I. oxyrinchus. As and Se concentrations were the

highest in M. manazo and T. scyllium, respectively. Zn and Sb were the highest in

C. umbratile. For Hg, I. oxyrinchus had the highest concentration (0.27 mg/kg)

followed by C. obscurus, C. brachyurus, C. umbratile, T. scyllium, and M. manazo.

The Cd concentration was under the DL (0.0001 mg/kg) in all 25 sharks, similar

to previous studies showing that most of the absorbed Cd were stored in the liver

and that only a small amount was stored in the muscle in sharks [12, 13].

Correlations between heavy metal concentrations and biological information

After the Box-Cox transformation, all the data satisfied the multivariate

normality assumption and homogeneity of variance-covariance matrix assumption,

except for only 6 cases (Table 3). Even though a few samples tested non-normal

or had a slight inequality of variance, MANOVA test could be performed since it

is fairly robust for deviations from multivariate normality and homogeneity of

variance-covariance.

19

MANOVA of Fe, Cu, Zn, As, Se, Hg, and MeHg concentrations for the 25

sharks showed that the concentrations of these heavy metals were significantly

associated with species (multivariate significance = 0.024; Table 8). Specifically,

Zn and As concentrations were significantly correlated with species.

In the case of As, it showed a significant correlation not only with species

but also with habitat and family (Fig 2A, 2B). The concentration of As differed

between benthic and pelagic sharks according to habitat-based classification. T.

scyllium, M. manazo, and C. umbratile are benthic sharks, whereas C. brachyurus,

C. obscurus, and I. oxyrinchus are pelagic sharks (Fig 2B). As concentrations

were significantly higher in benthic sharks (p < 0.05), and the pattern was similar

to that of a previous study showing higher concentrations of As in bottom-feeding

fish than in other bony fish [22]. Difference in As concentration was also found

according to phylogenetic families, i.e., Carcharhinidae (C. brachyurus and C.

obscurus), Isuridae (I. oxyrinchus), and Triakidae (T. scyllium and M. manazo; Fig

2A), presenting significantly different concentrations (p < 0.05) among the three

groups. In particular, As was the highest in Triakidae. Since Triakidae sharks

20

studied in this study are all benthic sharks, these results can be interpreted in the

same context.

In the case of Zn, the concentration was the highest in Carcharhinidae

sharks (Fig 2A). The concentration decreased in the order of Carcharhinidae,

Isuridae, and Triakidae, and this resembles the pattern of Zn concentration

appearing higher in pelagic sharks than in benthic sharks. This is also due to the

fact that Carcharhinidae and Isuridae are pelagic sharks.

Other heavy metals also showed concentration differences according to

habitat. In the case of Hg, the average concentration in benthic sharks was 0.14

mg/kg, which was 44% lower than that in pelagic sharks (0.25 mg/kg). The MeHg

concentration in benthic sharks (0.08 mg/kg) was 53% lower than that in pelagic

sharks (0.17 mg/kg). This is consistent with a previous study showing different

Hg and MeHg accumulation pattern between pelagic and benthic food chains [37].

Additionally, the average concentrations of Cu, Zn, Sn, and Pb were higher in

pelagic sharks, whereas the average concentrations of Fe and As were higher in

benthic sharks.

21

In the MVR analysis to confirm the correlation between continuous

variables (age, BW, girth, and TBL) and heavy metal concentrations, Hg and

MeHg were found to be significantly and positively correlated with BW, girth, and

TBL (Table 9, Fig 3A, 3B, 3F, 3G, 3J, 3K). A significant positive correlation also

existed between Zn and girth, Fe and girth, and Fe and TBL. The correlation

between Zn and girth is comparable with that of a previous study; as in the case of

the teleost fish Poecilia reticulata, it is known that Zn concentration is maintained

at a certain level in the body of these fish [38].

Se showed a significant correlation with girth and TBL, and was the only

heavy metal that showed a negative correlation (Fig 3E, 3I). This pattern is

different from those of previous studies showing that Se has positive correlation

with TBL in some tropical fishes or even that Se does not have any correlation

with TBL in various fish species including shortfin mako [39, 40].

Correlations between heavy metal concentrations and biological information in C.

brachyurus

22

The results of MANOVA in C. brachyurus (n = 15) showed that BW and

TBL are significantly correlated with both Hg and MeHg (p < 0.05; Table 8, Fig

3L, 3M, 3N, 3O). To find a regression model that explains the correlations better,

adjusted R2 values of polynomial and linear regressions were compared, and

polynomial regression was concluded as a more, well-fitting model (Fig 4A, 4B).

According to this regression model, Hg concentration in this species decreases

from birth to a certain point in their life, and then increases with time. This model

gives strength to the existence of growth dilution of Hg in C. brachyurus. The

slight decrease in Hg concentration at the early stage of growth can be deduced

from the effect of growth dilution in young individuals [17, 41]. Polynomial

regression of Hg concentrations according to girth and age also demonstrated

positive relationships (Fig 4D, 4E).

Based on this information, we speculated that Hg and MeHg

concentrations were all under the regulatory maximum limits because of the

sampled sharks’ ages and sizes. Prediction intervals of the regulatory maximum

limit of Hg (0.4 mg/kg) calculated using the regression lines of BW and TBL were

23

as follows: BW, 25.3–31.4 kg; TBL, 130.8–174.0 kg. These two criteria are easy

to confirm in the field and can be checked before a shark is sold at a local market

in order to avoid consumption of shark meat having Hg concentrations over the

limit value.

Hg and MeHg concentrations showed strong positive linear correlations

(Pearson’s r = 0.990; Fig 4C), and the average MeHg/Hg ratio was 61.5% ± 8.3%

(min–max: 46.3–83.3%), similar to previous reports [18, 19]. I. oxyrinchus had

the highest MeHg/Hg ratio (66.6%), and T. scyllium had the lowest MeHg/Hg

ratio (55.8%).

Heavy metal concentration differences according to sex in C. brachyurus

were analyzed. Fe, Cu, and Hg were high in males, whereas Zn, As, and Se were

high in females. In particular, Cu and As showed significant differences (p < 0.1;

Fig 4F). It is known that Cu concentration in some teleost fish is higher in females

than in males due to the metabolic activity difference, and a similar mechanism

can be considered for C. brachyurus in further studies [42].

By taking a closer look at the Cu concentration values, SNU-MO-0012

24

was a particularly noticeable case. This shark was by-caught right after birth,

having the umbilical cord still attached and its intestine filled with meconium only.

This shark’s Cu concentration was obviously the highest among all C. brachyurus,

and the cumulative percentage was 99.9%. Although the reason for this high Cu

concentration in younger individuals is unknown, this phenomenon is similar to

that observed in humans. Indeed, human babies show peak Cu concentrations

right after birth, with a subsequent decrease to one-tenth the initial level beginning

after 13 weeks of age [43, 44]. Consistent with this, the Cu concentration in SNU-

MO-0012 (8.91 mg/kg) was almost 10 times that of the other 14 C. brachyurus

sharks (0.92 mg/kg). This tendency was consistent with a previous study showing

that Cu concentration decreases during a shark’s growth period and increases

again after their maturation [8, 45]. A similar tendency was also found in marine

mammals whose Cu concentrations decreased with their growth [46, 47].

Pb concentrations showed a positive linear relationship with weight

(Pearson’s r = 0.729, p < 0.05), as confirmed by linear regression analysis. A

positive relationship was also observed for all 25 sharks (Pearson’s r = 0.578, p <

25

0.05), in contrast to previous studies demonstrating that there was a negative

relationship between fish body weight or size and Pb concentration [48, 49].

The correlation matrix of the 11 heavy metals in C. brachyurus showed

significant relationships between Fe and Cr (Pearson’s r = 0.538, p < 0.01), Sn and

Pb (Pearson’s r = 0.437, p < 0.05), and Hg and Pb (Pearson’s r = 0.752, p < 0.001).

However, the reason for these correlations is still unknown. In contrast, unlike the

known positive correlation between Se and Hg concentration in fish muscle, Se

and Hg concentrations in C. brachyurus were not significantly correlated

(Pearson’s r = 0.04) [14, 50]. This pattern did not change when the correlation was

analyzed with all the 25 sharks collected (Pearson’s r = 0.02).

26

Discussion

Regulatory limit of heavy metal concentration

Heavy metal concentration is one of the important criteria that should be

confirmed for food safety. Various institutions and governments have suggested

regulatory maximum limits for different heavy metals in fish (or predatory fish)

meat. To confirm the safety of shark meat as a food product, heavy metal

concentrations from this study were compared with the regulatory maximum

limits from eight international regulatory bodies and one domestic (Korean) food

regulatory body (Table 10).

The regulatory limits of As, Cd, Sn, Sb, Pb, Hg, and MeHg concentrations

are directly compared in Table 10. Because Hg and MeHg concentrations are

important criteria for determining food safety, various regulatory bodies have

presented maximum regulatory levels, and most have reported 1 mg/kg as the

limit value [51-57]; however, the Japanese Health Authority has suggested stricter

reference value, allowing up to 0.4 mg/kg total Hg and 0.3 mg/kg MeHg [58].

27

Even with the most stringent standards, the average concentration of Hg and

MeHg in all 25 sharks was below the limit value. Almost all 25 sharks showed

lower concentrations of Hg and MeHg than the regulatory maximum limits,

except for two sharks that had the longest TBL and largest BW (SNU-MO-0006

and SNU-MO-0001). These two sharks had Hg concentrations of 268% and 102%

of the maximum limit concentrations, respectively, and both were C. brachyurus

species. Furthermore, only one shark (SNU-MO-0006), which had the longest

TBL and largest BW, exceeded the limit for the MeHg concentration (297% of the

limit value). In contrast, the As concentrations of all shark species, except for I.

oxyrinchus, exceeded the regulatory limit value. The average concentration was

less than the regulatory limit in I. oxyrinchus, but was fairly close to the limit

value. The Average As concentration in all 25 sharks was 7.71 mg/kg, which was

more than twice the regulatory level. The concentrations of other heavy metals

(Sb, Pb, Cd, and Sn) were within the safe range in all 25 sharks.

For Cr, Cu, Zn, and Se, the regulatory maximum limits were indicated in

mg/day; thus, the measured concentrations were converted to mean daily intake

28

amounts of heavy metals from shark meat (mg/day) by multiplying the average

amount of seafood ingested by Koreans (79.6 g/day) for each concentration, and

these values were then compared with the regulatory limits [59]. The

concentrations of all four heavy metals were under the regulatory maximum limits

for all shark species (Table 10).

Food safety of shark meat and the need for shark meat consumption guideline

Shark meat is concluded as not safe to be used as a food source due to the

Hg, MeHg, and As concentrations, even though concentrations of Cr, Cu, Zn, Se,

Cd, Sn, Sb, and Pb were lower than the regulatory limits (Table 10).

Hg toxicity due to overexposure results in serious clinical problems

including neurotoxicity, cardiovascular toxicity, and embryotoxicity in pregnancy,

of which Minamata disease is representative. MeHg is one of the organic Hg

species and it causes secondary structural change to DNA and RNA and also

protein structural change by binding to thiol groups [23]. Since MeHg has a good

29

absorption rate (90%) and long retention time, caution should be exercised when

consuming fish meat. According to various regulations for food safety, as

discussed above, almost every shark showed Hg and MeHg concentration

belonging to the safety zone, except for 2 and 1 individual sharks, respectively.

Even though Hg concentration itself seldom exceeded the regulatory

limits in this study, shark meat consumption should still be considered carefully

due to various reasons. Previous studies found that combined exposure to Hg and

other heavy metals including Al, Cu, Pb, Cd, and Mn poses a synergetic health

risk and that shark meat consumption can easily expose an individual to this

health risk [61, 62]. Furthermore, excess Hg concentration can be predicted in a

copper shark with over 25 kg BW and 130 cm TBL based on the positive

relationship found in this study. Since meat from a copper shark over the predicted

size will not be appropriate as a food source, the predicted value can be used as a

guideline for consumers to choose more safe copper shark meat and for using

different shark species as meat. Since Hg concentration also differs from organ to

organ, and shark liver, fin, and intestine are also used as food sources, guidelines

30

for different organs should also be prepared [9, 20].

The need for a more detailed guideline for shark meat safety also exists

for As, which showed excess concentration in almost every shark examined.

Arsenic toxicity results in various manifestations such as skin lesions, chronic

lung diseases, liver diseases, and vascular diseases. In addition, since As inhibits

mitochondrial respiration, which leads to oxidative stress and successive cell

injury, death, and even cancer, its excessive accumulation must be avoided [63].

In this study, As showed significantly higher concentration in benthic sharks than

in pelagic sharks, and in fact, all shark species except I. oxyrinchus showed

regulatory limit exceedance. As the concentration of As varies according to the

habitat and species, it is necessary to study the As concentration on the basis of a

more diversified shark species and propose a guideline to prevent excessive

accumulation through shark meat ingestion.

31

Fig 1. Sampling location in Jeju Island. Nineteen sharks were sampled from the Moseulpo fish market (30,

Hamohanggu-ro, Daejeong-eup, Seogwipo-si, Jeju-do 63506, Republic of Korea) and 6 sharks were from the Hallim fish

market (141-3, Hallimhaean-ro, Hallim-eup, Jeju-si, Jeju-do 63032, Republic of Korea).

32

Fig 2. Average heavy metal concentration comparison by family and habitat. Data under the DL were substituted

with half the DL. Error bars indicate standard errors. *: p < 0.05; Pelagic sharks: C. brachyurus, C. obscurus, I.

oxyrinchus; benthic sharks: T. scyllium, M. manazo, C. umbratile. Carcharhinidae: C. brachyurus, C. obscurus;

Triakidae: T. scyllium, M. manazo; Isuridae: I. oxyrinchus.

33

Fig 3. Multivariate regression graph. A – K: Multivariate linear regression

between BW, TBL, girth and heavy metal concentrations in every 25 sharks. L –

O: Multivariate linear regression between BW, TBL and heavy metal

concentrations in C. brachyurus. All A – O showed significant correlation

(p<0.05).

34

Fig 4. Heavy metal concentration analysis in C. brachyurus. A, B, D, E: Correlation between Hg concentration and

body weight (BW), total body length (TBL), girth, and age in C. brachyurus (p < 0.05 in all four cases). Blue lines show

the 95% prediction interval for the regression line, and blue dotted lines indicate the regulatory maximum limits of Hg

and MeHg (0.4 and 0.3 mg/kg, respectively). C: Correlation between the concentrations of Hg and MeHg in C.

brachyurus (Pearson’s r = 0.990, p < 0.05). F: Average heavy metal concentration comparison between male and female

C. brachyurus. Error bars indicate standard errors, and the spots beyond the error bars indicate significance at p < 0.1.

35

Table 1. Shapiro-Wilk test & Bartlett’s test results before transformation. Normality assumption and homogeneity

of variance-covariance assumption are not all satisfied. All values were rounded to the fourth decimal place.

Tests Variables Fe Cu Zn As Se Hg MeHg

All sharks

Shapiro-Wilk test

Species 0.0471 0.0002 0.0003 0.0057 7.53e-08 1.36e-06 1.17e-07

Sex 0.0019 4.30e-07 0.0110 0.0326 3.79e-07 3.70e-06 4.89e-07

Habitat 0.0028 3.95e-06 9.75e-05 0.0792 6.59e-08 1.21e-06 9.85e-08

Bartlett’s test

Species 0.3350 0.0116 0.0173 0.0550 0.0132 0.0201 0.0038

Sex 0.8099 0.7985 0.0278 0.9001 0.0001 0.0018 0.0001

Habitat 0.4903 0.0003 0.8884 0.1638 0.0171 0.0013 0.0001

Carcharhinus brachyurus

Shapiro-Wilk test Sex 0.0069 0.0017 0.1142 0.3260 5.66e-05 0.0068 0.0010

Bartlett’s test Sex 0.6372 3.06e-07 0.0074 0.0395 0.0170 0.0033 0.0004

36

Table 2. Estimated transformation parameters for multivariate Box-Cox transformation. Variables were replaced

by a simple power transformation after estimation.

Variables Fe Cu Zn As Se Hg MeHg

All sharks

Species 0.25 0.25 -0.5 0.25 0 -0.25 -0.25

Sex 0.25 0.25 -0.25 0.25 0 -0.25 -0.25

Habitat 0.25 0.25 -0.25 0.25 0 -0.25 -0.25

Copper shark

Sex 0.25 0.25 -0.25 -0.5 -0.25 -0.25 -0.25

37

Table 3. Shapiro-Wilk test & Bartlett’s test results after transformation. Normality assumption and homogeneity of

variance-covariance assumption are satisfied. Even though a few samples tested as non-normal or with slightly inequality

of variance, MANOVA is fairly robust to deviations from normality and homoscedasticity. All values were rounded to the

fourth decimal place.

Tests Variables Fe Cu Zn As Se Hg MeHg

All sharks

Shapiro-Wilk test

Species 0.3826 0.1606 0.7947 0.0834 0.1242 0.5626 0.1920

Sex 0.0552 0.0146 0.7230 0.8371 0.3933 0.7866 0.8764

Habitat 0.2087 0.0543 0.4147 0.7111 0.2549 0.8392 0.7926

Bartlett’s test

Species 0.5023 0.2628 0.3190 0.0747 0.0709 0.2261 0.0977

Sex 0.9543 0.2969 0.2167 0.5445 0.2032 0.5087 0.5299

Habitat 0.2759 0.0427 0.5710 0.9488 0.0982 0.0918 0.0156

Carcharhinus brachyurus

Shapiro-Wilk test Sex 0.4399 0.0189 0.7540 0.2873 0.8022 0.7862 0.8913

Bartlett’s test Sex 0.1741 0.0001 0.0464 0.3743 0.5555 0.3750 0.3957

38

Table 4. Mardia’s multivariate normality (MVN) test & Henze-Zirkler’s MVN test after transformation.

Multivariate normality is satisfied. All values were rounded to the third decimal place.

VariablesMardia’s MVN test (result (p value)) Henze-Zirkler’s MVN test

Mardia skewness Mardia kurtosis

All sharks

Species Yes (0.284) Yes (0.963) Yes (0.401)

Sex Yes (0.705) Yes (0.365) Yes (0.367)

Habitat Yes (0.951) Yes (0.183) Yes (0.669)

Carcharhinus brachyurus

Sex Yes (0.772) Yes (0.093) Yes (0.293)

39

Table 5. Total body length, body weight, age, and sampling location of sampled sharks. Data not available were

excluded from the mean and standard deviation calculation. Missing value proportions of total body length, body weight,

and age are 4%, 32%, and 24%, respectively. Sharks on the first year after birth were counted as 0 years old. All average

and standard deviation values were rounded to the second decimal place.

Species nMean ± SD (Min-Max)

Sampling locationTotal body length (cm) Body weight (kg) Age (yr)

Carcharhinus brachyurus 15124.02 ± 32.70

(68-190)

15.33 ± 12.06

(4.5-45)

2.64 ± 1.86

(0-7)Moseulpo fish market

Carcharhinus obscurus 2116.00 ± 2.83

(114-118)

8.45 ± 0.78

(7.9-9)

0.0 ± 0.0

(0-0)Moseulpo fish market

Isurus oxyrinchus 2123.50 ± 19.09

(110-137)

12.60 ± 7.21

(7.5-17.7)

1.0 ± 0.0

(1-1)Moseulpo fish market

Triakis scyllium 370.00 ± 3.77

(66.5-74)

1.70 ± 0.00

(1.7-1.7)

2.0 ± 0.0

(2-2)Hallim fish market

Mustelus manazo 268.00 ± 22.63

(84-52)

0.50 ± 0.00

(0.5-0.5)

2.0 ± 0.0

(2-2)Hallim fish market

Cephaloscyllium

umbratile1 63.0 - - Hallim fish market

40

Table 6. Heavy metal concentrations and biological data of the sampled sharks. Concentrations under detection limit

(DL) are marked as <DL. All concentrations are WW concentrations. M: Moseulpo; H: Hallim; F: female; M: male; P:

pelagic; B: benthic

ID Species

Loca

-tion

Heavy metal concentration (mg/kg, WW) Biologic data

Cr Fe Cu Zn As Se Cd Sn Sb Pb THg MeHg Age

TBL

(cm)

BW

(kg)

Girth

(cm)

Sex

Habi-

tat

SNU-

MO-0001

Carcharhinus

brachyurus

M < DL 26.7147 0.5546 22.8517 15.1225 0.5799 < DL < DL < DL 0.0761 0.4089 0.2425 4 147 - 67 F P

SNU-

MO-0002

Carcharhinus

brachyurus

M < DL 4.6736 0.3288 4.4662 11.1706 3.2649 < DL < DL < DL 0.0835 0.2944 0.2026 0 100.5 - 42 F P

SNU-

MO-0003

Carcharhinus

brachyurus

M < DL < DL 0.8858 7.1427 5.4523 0.6352 < DL 0.2082 < DL 0.06 0.0665 0.0337 2 87 - 38 F P

SNU-

MO-0004

Carcharhinus

brachyurus

M < DL 8.2137 0.4604 13.7624 10.4339 0.519 < DL 0.0351 < DL 0.0195 0.0963 0.0598 2 114 9 50.5 F P

SNU-

MO-0005

Carcharhinus

brachyurus

M < DL 0.9725 0.5482 8.7583 10.3842 0.576 < DL < DL < DL 0.0217 0.1105 0.0692 3 109.8 8.5 45 F P

SNU-

MO-0006

Carcharhinus

brachyurus

M 0.1648 11.1677 1.0163 7.3183 6.7375 0.31 < DL 0.0314 < DL 0.1962 1.0706 0.8921 - 190 45 - M P

SNU-

MO-0007

Isurus

oxyrinchus

M < DL 12.2207 7.7498 4.3619 1.3043 0.4194 < DL 0.0165 < DL 0.0429 0.2215 0.1322 1 110 7.5 54 F P

41

SNU-

MO-0008

Carcharhinus

obscurus

M < DL 2.2459 0.1323 5.3205 8.0156 0.3169 < DL < DL < DL 0.0383 0.2713 0.1639 0 114 7.9 52 M P

SNU-

MO-0009

Carcharhinus

obscurus

M 0.7492 23.0075 5.3178 10.9969 7.6799 0.3731 < DL 0.1822 < DL 0.1293 0.2400 0.1667 0 118 9 54 F P

SNU-

MO-0010

Isurus

oxyrinchus

M < DL 14.7325 1.1469 2.0948 2.8112 0.2931 < DL 0.1302 < DL 0.0687 0.3266 0.2399 - 137 17.7 - M P

SNU-

MO-0011

Cephaloscylliu

m umbratile

H 0.1588 5.657 0.5731 13.5636 8.0886 0.2401 < DL < DL 0.0774 0.0697 0.1806 0.1094 - 63 - - M B

SNU-

MO-0012

Carcharhinus

brachyurus

M 0.0032 2.9276 8.9158 4.4975 4.0259 0.7248 < DL 0.0063 0.0443 0.0063 0.1720 0.1111 0 68 1.65 25 M P

SNU-

MO-0013

Carcharhinus

brachyurus

M 0.083 3.2612 1.0627 13.8818 6.9973 0.2159 < DL 0.0199 0.0465 0.0166 0.1491 0.0748 3 143 19.5 60 F P

SNU-

MO-0014

Carcharhinus

brachyurus

M < DL 6.9166 < DL 4.157 4.5788 0.1288 < DL 0.0354 < DL 0.0354 0.0983 0.0628 1 92 4.5 - M P

SNU-

MO-0015

Carcharhinus

brachyurus

M 0.011 20.6689 3.6917 5.0847 5.8473 0.1613 < DL 0.044 < DL 0.0403 0.1168 0.0620 2 118 9.2 47.5 M P

SNU-

MO-0016

Carcharhinus

brachyurus

M 0.103 3.1122 0.5187 2.3799 4.9429 0.0687 < DL < DL < DL 0.0038 0.1166 0.072 2 121 12 51 F P

SNU-

MO-0017

Carcharhinus

brachyurus

M < DL 7.3542 0.7387 3.6387 3.924 0.0841 < DL 0.0037 < DL < DL 0.3739 0.208 7 165 26 69 M P

SNU-

MO-0018

Carcharhinus

brachyurus

M 0.0425 2.0243 0.2477 2.1765 4.4131 0.1557 < DL < DL < DL < DL 0.1532 0.102 3 140 16.6 56.5 F P

42

SNU-

MO-0019

Carcharhinus

brachyurus

M 0.5674 12.8523 1.1071 3.609 5.6972 0.2719 < DL < DL < DL < DL 0.2367 0.117 5 - - 57.5 - P

SNU-

MO-0020

Mustelus

manazo

H 0.1247 4.0576 0.6725 2.0363 15.0213 0.4496 < DL < DL < DL 0.0113 0.1481 0.089 2 84 - 25.5 F B

SNU-

MO-0021

Triakis

scyllium

H 0.7698 25.4804 0.2694 4.1184 8.718 0.7621 < DL < DL < DL 0.0231 0.1481 0.086 2 74 1.7 26.5 M B

SNU-

MO-0022

Mustelus

manazo

H 0.0138 < DL 0.1797 2.149 20.1565 0.3731 < DL < DL < DL < DL 0.0922 0.066 - 52 0.5 16.5 M B

SNU-

MO-0023

Carcharhinus

brachyurus

M 0.0676 12.3939 0.823 4.938 4.9718 0.0789 < DL < DL < DL 0.0188 0.2139 0.131 3 141 16.7 56 F P

SNU-

MO-0024

Triakis

scyllium

H 0.1701 3.3286 0.1314 2.9768 7.9794 0.4523 < DL < DL < DL 0.0232 0.1382 0.087 - 69.5 - 26 M B

SNU-

MO-0025

Triakis

scyllium

H < DL 1.1079 0.1443 3.1325 8.2584 0.7061 < DL < DL < DL < DL 0.1052 0.049 - 66.5 - 26.5 M B

43

Table 7. Average concentrations (mg/kg WW) of 11 metals and methyl mercury in six shark species. Values under

DL were substituted with half the value for statistical analysis. All values were rounded to the second decimal place.

Species nMetal concentration (Mean ± standard deviation)

Cr Fe Cu Zn As Se Cd Sn Sb Pb Hg MeHg

Carcharhinus

brachyurus15

0.07 ±

0.15

8.22 ±

7.54

1.39 ±

2.25

7.24 ±

5.62

6.98 ±

3.29

0.52 ±

0.79

0.00 ±

0.00

0.03 ±

0.05

0.01 ±

0.02

0.04 ±

0.05

0.25 ±

0.25

0.16 ±

0.21

Carcharhinus

obscurus2

0.37 ±

0.53

12.63 ±

14.68

2.73 ±

3.67

8.16 ±

4.01

7.85 ±

0.24

0.35 ±

0.04

0.00 ±

0.00

0.09 ±

0.13

0.00 ±

0.00

0.08 ±

0.06

0.26 ±

0.02

0.17 ±

0.00

Isurus

oxyrinchus2

0.00 ±

0.00

13.48 ±

1.78

4.45 ±

4.67

3.23 ±

1.60

2.06 ±

1.07

0.36 ±

0.09

0.00 ±

0.00

0.07 ±

0.08

0.00 ±

0.00

0.06 ±

0.02

0.27 ±

0.07

0.19 ±

0.08

Triakis

scyllium3

0.31 ±

0.40

9.97 ±

13.48

0.18 ±

0.08

3.41 ±

0.62

8.32 ±

0.37

0.64 ±

0.17

0.00 ±

0.00

0.00 ±

0.00

0.00 ±

0.00

0.02 ±

0.01

0.13 ±

0.02

0.07 ±

0.02

Mustelus

manazo2

0.07 ±

0.08

2.03 ±

2.87

0.43 ±

0.35

2.09 ±

0.08

17.59 ±

3.63

0.41 ±

0.05

0.00 ±

0.00

0.00 ±

0.00

0.00 ±

0.00

0.01 ±

0.01

0.12 ±

0.04

0.08 ±

0.02

Cephaloscylli-

um umbratile1 0.16 5.66 0.57 13.56 8.09 0.24 0.00 0.00 0.08 0.07 0.18 0.11

Total 250.12 ±

0.23

8.60 ±

8.08

1.49 ±

2.37

6.37 ±

5.06

7.71 ±

4.25

0.49 ±

0.62

0.00 ±

0.00

0.03 ±

0.06

0.01 ±

0.02

0.04 ±

0.05

0.21 ±

0.20

0.13 ±

0.16

44

Table 8. MANOVA test. Multivariate significance (MVS) was calculated by analyzing Fe, Cu, Zn, As, Se, Hg, and

MeHg based on the criteria of species, sex, and habitat of sharks. For C. brachyurus, MANOVA could be performed by

incorporating sex. All values were rounded to the third decimal place. ***: p < 0.001; **: p < 0.01; *: p < 0.05.

Variables MVSMetals

Fe Cu Zn As Se Hg MeHg

All sharks

Species 0.024* 0.346 0.261 0.045* 0.001*** 0.740 0.471 0.452

Sex 0.909 0.933 0.372 0.216 0.762 0.689 0.582 0.581

Habitat 0.240 0.459 0.208 0.234 0.016* 0.474 0.313 0.451

Carcharhinus brachyurus

Sex 0.289 0.264 0.395 0.452 0.069 0.489 0.334 0.337

45

Table 9. Multivariate regression (MVR). MVR was analyzed and p values were calculated on Fe, Cu, Zn, As, Se, Hg,

and MeHg on the criteria of age, BW, girth, and TBL. For C. brachyurus, MVR was also performed by incorporating the

same biological criteria. All values were rounded to the third decimal place. ***: p < 0.001; **: p < 0.01; *: p < 0.05.

Variables Fe Cu Zn As Se Hg MeHg

All sharks

Age 0.681 0.522 0.912 0.932 0.058 0.374 0.653

BW 0.400 0.966 0.617 0.435 0.140 0.001** 0.002**

Girth 0.032* 0.166 0.031* 0.091 0.029* 0.008** 0.017*

TBL 0.033* 0.478 0.276 0.151 0.043* 0.001*** 0.001**

Carcharhinus brachyurus

Age 0.384 0.837 0.954 0.718 0.123 0.122 0.230

BW 0.554 0.571 0.879 0.721 0.173 0.002** 0.005**

Girth 0.079 0.245 0.828 0.702 0.071 0.074 0.131

TBL 0.092 0.937 0.333 0.707 0.160 0.009** 0.011*

46

Table 10. Regulatory maximum limits of metals in shark meat. The regulatory maximum limits and mean heavy

metal concentrations of As, Cd, Sn, Sb, Pb, Hg, and MeHg are shown in mg/kg. For Cr, Cu, Zn, and Se, the regulatory

maximum limits and mean daily heavy metal intake amounts from shark meat are shown in mg/day. The regulatory limit

of Fe has not been specified. Red box: mean value exceeding the regulatory maximum limit. Yellow box: data that

exceed the regulatory maximum limit but the mean value does not. Green box: every data is under the regulatory

maximum limit. a: DOH [54]; b: DOH [53]; c: CODEX [52]; d: SCF [60]; e: FAO [56]; f: JECFA [57]; g: EU [55]; h:

KFDA [51]; i: UNEP [58].

SpeciesMetal concentration comparison with regulatory maximum limits

Cr Cu Zn As Se Cd Sn Sb Pb Hg MeHg

Regulatory

maximum limits0.25d 0.5d 25d 3b 0.3d

0.05~

1b,e~h50b 0.15b

0.2~

2a~c,e~h

0.4~

1e~g,i

0.3~

1a~c,h,i

Carcharhinus brachyurus 0.01 0.12 0.51 6.98 0.04 0.00 0.03 0.01 0.04 0.25 0.16

Carcharhinus obscurus 0.03 0.22 0.65 7.85 0.03 0.00 0.09 0.00 0.08 0.26 0.17

Isurus oxyrinchus 0.00 0.35 0.26 2.06 0.03 0.00 0.07 0.00 0.06 0.27 0.19

Triakis scyllium 0.02 0.01 0.27 8.32 0.05 0.00 0.00 0.00 0.02 0.13 0.07

Mustelus manazo 0.01 0.03 0.17 17.59 0.03 0.00 0.00 0.00 0.01 0.12 0.08

Cephaloscyllium umbratile 0.01 0.05 1.08 8.09 0.02 0.00 0.00 0.08 0.07 0.18 0.11

47

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55

Summary

Sharks are vulnerable to bioaccumulation of various substances including

heavy metals since they are apex predators. In this study, five shark species

showed As concentrations that exceeded the regulatory maximum limit, whereas

almost all Hg and MeHg concentrations were below the regulatory limits. This

was likely because the sampled sharks were juveniles, and Hg and MeHg levels

showed strong positive correlations with TBL, BW, and age. Because a C.

brachyurus individual of over 25 kg in BW or 130 cm in TBL is likely to have Hg

concentrations exceeding the regulatory maximum limit, avoiding consumption of

such adult C. brachyurus can reduce the risk of Hg toxicity. The average

concentrations of other heavy metals (Cr, Fe, Cu, Zn, Se, Cd, Sn, Sb, and Pb)

were all in the safe range. Despite this, caution should be exercised when

consuming shark meat owing to the As, Hg and MeHg concentrations. Since shark

meat is being consumed globally, especially in Asian countries, detailed

guidelines should further be prepared for safe consumption based on extensive

studies of heavy metals in different shark species.

56

국문 초록

제주도 연근해 상어육의 중금속 농도

및 식품안전성

김 상 화

서울대학교 대학원 수의학과 수의병인생물학 및 예방수의학

전공

(지도교수: 박 세 창)

상어고기는 아시아를 필두로 하여 전 세계 다양한 국가에서

식용자원으로 이용되어 왔다. 그러나, 이들은 해양생태계의

최상위층에 존재하는 포식자이기 때문에 중금속 생물축적에 취약하다.

인간의 소비를 위해 연간 1억마리 이상의 상어가 포획되고 있는

만큼, 상어고기의 안전성 판단은 중요한 과제이다. 본 연구에서는

한국 제주도 연근해에 서식하는 6종 (이주성 상어 3종: Carcharhinus

brachyurus, Carcharhinus obscurus, Isurus oxyrinchus; 정착성 상어 3종:

Triakis scyllium, Mustelus manazo, Cephaloscyllium umbratile) 상어

57

근육조직의 중금속 농도를 분석하였다. 총 11종의 중금속 (Cr, Fe, Cu,

Zn, As, Se, Cd, Sn, Sb, Pb, Hg) 및 MeHg의 농도를 ICP-MS, DMA, GC

및 CVAFS를 이용하여 확인하고 국제식품규격위원회를 포함하여

다양한 기관에서 제시하고 있는 최대규제치 값과 비교하였다. 그

결과 As를 제외한 모든 중금속의 평균 농도는 규제치 이하인 것으로

나타났다. 그 중 Hg와 MeHg는 체장, 체중 및 나이와 유의미한 양의

상관관계를 보였으며, C. brachyurus 종의 경우 체장 130cm, 체중

25kg 이상인 개체에서는 높은 확률로 Hg 농도가 규제치를 초과할

것으로 계산되었다. 본 연구 결과 상어고기를 소비할 경우

소비자들은 높은 농도의 As에 노출될 수 있으며, 예측된 크기보다

큰 C. brachyurus유래 고기를 소비하는 경우 또한 높은 농도의

Hg에 노출될 수 있음이 확인되었다. 추가적인 연구를 통해 다양한

상어고기의 소비 시 적용할 세밀한 규제 및 가이드라인을 설정해야

할 필요성이 제시된다.

핵심어: 상어고기, 중금속, 식품안전성, 생물축적, Carcharhinus

brachyurus

학번: 2016-26230

58

List of articles

2019 Published

1. Sang Wha Kim, Se Jin Han, Yonggab Kim, Jin Woo Jun, Sib Sankar Giri,

Cheng Chi, Saekil Yun, Hyoun Joong Kim, Sang Guen Kim, Jeong Woo Kang,

Jun Kwon, Woo Taek Oh, Jehyun Cha, Seunghee Han, Byeong Chun Lee,

Taesung Park, Byung Yeop Kim, and Se Chang Park* (2019). Heavy metal

accumulation in and food safety of shark meat from Jeju Island, Republic of

Korea. PLOS ONE. 10.1371/journal.pone.0212410

2. Sang Guen Kim, Jin Woo Jun, Sib Sankar Giri, Saekil Yun, Hyoun Joong Kim,

Sang Wha Kim, Jeong Woo Kang, Se Jin Han, Dalsang Jeong, and Se Chang

Park*. Isolation and characterization of pVa-21, a giant bacteriophage with

anti-biofilm potential against Vibrio alginolyticus. Scientific Reports.

3. Hyoun Joong Kim, Jin Woo Jun, Sib Sankar Giri, Saekil Yun, Sang Guen Kim,

Sang Wha Kim, Jeong Woo Kang, Se Jin Han, Jun Kwon, Woo Taek Oh,

Dalsang Jeong, Hyoung Bae Jeon, and Se Chang Park*. Mass mortality in

Korean bay scallop (Argopecten irradians) caused by Ostreid Herpesvirus-1.

Transboundary and Emerging Disease.

2018 Published

1. Sang Wha Kim, Jin Woo Jun, Sib Sankar Giri, Cheng Chi, Saekil Yun,

Hyoun Joong Kim, Sang Guen Kim, Jeong Woo Kang, and Se Chang Park*

(2018). First Report of Carp Edema Virus Infection of Koi (Cyprinus carpio

haematopterus) in the Republic of Korea. Transboundary and Emerging

Diseases. 10.1111/tbed.12782.

59

2. Hyoun Joong Kim, Jin Woo Jun, Sib Sankar Giri, Cheng Chi, Saekil Yun,

Sang Guen Kim, Sang Wha Kim, Jeong Woo Kang, Se Jin Han, Se Chang

Park* (2018). Complete genome sequence of bacteriophage pVco-5 that

infects Vibrio corallilyticus, which causes bacillary necrosis in Pacific oyster

(Crassostrea gigas) larvae. Genome Announcements. 6(2): e01143-17.

3. Sib Sankar Giri, Jin Woo Jun, Saekil Yun, Hyoun Joong Kim, Sang Guen Kim,

Jeong Woo Kang, Sang Wha Kim, Se Jin Han, Se Chang Park*, and V.

Sukumaran* (2018). Characterisation of Lactic Acid Bacteria Isolated from

the Gut of Cyprinus carpio That May Be Effective Against Lead Toxicity.

Probiotics and Antimicrobial Proteins. 10.1007/s12602-017-9367-6.

4. Sib Sankar Giri, Saekil Yun, Jin Woo Jun, Hyoun Joong Kim, Sang Guen Kim,

Jeong Woo Kang, Sang Wha Kim, Se Jin Han, V. Sukumaran, Se Chang

Park* (2018). Therapeutic Effect of Intestinal Autochthonous Actobacillus

reuteri P16 Against Waterborne Lead Toxicity in Cyprinus carpio. Frontiers in

Immunology. 10.3389/fimmu.2018.01824.

5. Cheng Chi, Sib Sankar Giri, Jin Woo Jun, Sang Wha Kim, Hyoun Joong Kim,

Jeong Woo Kang, Se Chang Park* (2018). Detoxification- and Immune-

Related Transcriptomic Analysis of Gills from Bay Scallops (Argopecten

irradians) in Response to Algal Toxin Okadaic Acid. Toxins.

10.3390/toxins10080308.

6. Jin Woo Jun, Sib Sankar Giri, Hyoun Joong Kim, Saekil Yun, Cheng Chi,

Sang Guen Kim, Sang Wha Kim, Jeong Woo Kang, Se Jin Han, Jun Kwon,

Woo Taek Oh, Dalsang Jeong, Se Chang Park* (2018). Identification and

phylogenetic characterization of Nybelinia surmenicola (Cestoda:

Trypanorhyncha) from squids (Todarodes pacificus) from East Sea, Republic

of Korea. Journal of Preventive Veterinary Medicien. 42(3), 112-116.

60

7. Cheng Chi, Sib Sankar Giri, Jin Woo Jun, Hyoun Joong Kim, Sang Wha Kim,

Jeong Woo Kang, Se Chang Park* (2018). Detoxification and Immune

Transcriptomic Response of the Gill Tissue of Bay Scallop (Argopecten

irradians) Following Exposure to the Algicide Palmitoleic Acid. Biomolecules.

2018, 8(4), 139; doi: 10.3390/biom8040139.

8. Saekil Yun, Jin Woo Jun, Sib Sankar Giri, Hyoun Joong Kim, Cheng Chi,

Sang Geun, Kim, Sang Wha Kim, Jung Woo Kang, Se Jin Han, Jun Kwon,

Woo Taek Oh, Se Chang Park* (2018). Immunostimulation of Cyprinus

carpio using phage lysate of Aeromonas hydrophila. Fish and Shellfish

Immunology. 10.1016/j.fsi.2018.11.076

2017 Published

1. Jin Woo Jun, Sib Sankar Giri, Hyoun Joong Kim, Cheng Chi, Saekil Yun,

Sang Guen Kim, Sang Wha Kim, Jeong Woo Kang, and Se Chang Park*

(2017). Compete Genome Sequence of the Novel Bacteriophage pSco-10

Infecting Staphylococcus cohnii. Genome Announcements. 5(47): e01032-17.

2. Jusun Hwang, Kyunglee Lee, Daniel Walsh, Sang Wha Kim, Jonathan M.

Sleeman, Hang Lee* (2017). Semi-quantitative assessment of disease risks at

the human, livestock, wildlife interface for the Republic of Korea using a

nationwide survey of experts: A model for other countries. Transboundary and

Emerging Diseases. 10.1111/tbed.12705.

3. Ji Hyung Kim, Young Ran Lee, Jeong Rack Koh, Jin Woo Jun, Sib S‑ ‑ ankar

Giri, Hyoun Joong Kim, Cheng Chi, Saekil Yun, Sang Geun Kim, Sang Wha

Kim, Hye Kwon Kim, Dae Gwin Jeong, Se Chang Park* (2017). Complete

mitochondrial genome of the beluga whale Delphinapterus leucas (Cetacea:

Monodontidae). Conservation Genetics Resources. 10.1007/s12686-017-0705-5.

61

4. Saekil Yun, Young-Ran Lee, Sib Sankar Giri, Hyoun Joong Kim, Cheng Chi,

Sang Guen Kim, Sang Wha Kim, Jin Woo Jun, Se Chang Park* (2017).

Isolation of a zoonotic pathogen Aeromonas hydrophila from freshwater

stingray (Potamotrygon motoro) kept in a Korean aquarium with ricefish

(Oryzias latipes). Korean Journal of Veterinary Research. 57(1):67-69.

5. Cheng Chi, Sib Sankar Giri, Jin Woo Jun, Hyoun Joong Kim, Sang Wha Kim,

Saekil Yun, Se Chang Park* (2017). Effects of algal toxin okadaic acid on the

non-specific immune and antioxidant response of bay scallop (Argopecten

irradians). Fish and Shellfish Immunology. 65: 111-117.

6. Sang Guen Kim, Jin Woo Jun, Sib Sankar Giri, Saekil Yun, Hyoun Joong Kim,

Cheng Chi, Sang Wha Kim, Se Chang Park* (2017). Complete Genome

Sequence of Staphylococcus aureus Bacteriophage pSa-3. Genome

Announcement. 5(21): e00182-17.

7. Hyoun Joong Kim, Ji Hyung Kim, Jin Woo Jun, Sib Sankar Giri, Cheng Chi,

Saekil Yun, Sang Guen Kim, Sang Wha Kim, Jeong Woo Kang, Dae Gwin

Jeong, Se Chang Park* (2017). Complete Genome Sequence of Vibrio

coralliilyticus 58, Isolated from Pacific Oyster (Crassostrea gigas) Larvae.

Genome Announcement. 5(23): e00437-17.

8. Saekil Yun, Jin Woo Jun, Sib Sankar Giri, Hyoun Joong Kim, Cheng Chi,

Sang Geun Kim, Sang Wha Kim, Se Chang Park* (2017). Efficacy of PLGA

microparticle-encapsulated formalin-killed Aeromonas hydrophila cells as a

single-shot vaccine against A. hydrophila infection. Vaccine. 35(32): 3959-

3965.

62

List of conference

2018

1. Hyoun Joong Kim, Jin Woo Jun, Sib Sankar Giri, Seakil Yun, Sang Guen Kim,

Sang Wha Kim, Jeong Woo Kang, Se Jin Han, Jun Kwon, Woo Taek Oh and

Se Chang Park, Application of Bacteriophages to Prevent Mass mortality

Caused by Vibrio coralliilyticus in Pacific oyster (Crassostrea gigas) Larvae.

3rd Aquaculture conference. Qingdao, China, 2018.

2. Jeong Woo Kang, Jin Woo Jun, Sib Sankar Giri, Seakil Yun, Hyoun Joong

Kim, Sang Guen Kim, Sang Wha Kim, Se Jin Han, Jun Kwon, Woo Taek Oh

and Se Chang Park, Superiority of PLGA microparticle-encapsuled formalin-

killed cell vaccine to conventional formalin-killed cell vaccine in protection

against Streptococcus parauberis infection in olive flounder (Paralichthys

olivaceus). 3rd Aquaculture conference. Qingdao, China, 2018.

3. Seakil Yun, Jin Woo Jun, Sib Sankar Giri, Hyoun Joong Kim, Sang Guen Kim,

Sang Wha Kim, Jeong Woo Kang, Se Jin Han, Jun Kwon, Woo Taek Oh and

Se Chang Park, Efficacy of Poly Lactic-co-glycolic Acid Microparticle

Vaccine using Bacteriophage Lysates of Bacteria for Bacterial Infection in

Fish. 3rd Aquaculture conference. Qingdao, China, 2018.

4. Se Jin Han, Jin Woo Jun, Sib Sankar Giri, Seakil Yun, Hyoun Joong Kim,

Sang Guen Kim, Sang Wha Kim, Jeong Woo Kang, Jun Kwon, Woo Taek Oh

and Se Chang Park, Candida manassasensis infection in digestive tracts of

Siberian sturgeon Acipenser baerii fingerlings. 3rd Aquaculture conference.

Qingdao, China, 2018.

63

2017

1. Sang Wha Kim, Jin Woo Jun, Sib Sankar Giri, Cheng Chi, Saekil Yun, Hyoun

Joong Kim, Sang Guen Kim, Jeong Woo Kang, Se Chang Park*. Detection

and Phylogenetic Analysis of Carp Edema Virus in Koi (Cyprinus carpio

haematopterus) in the Republic of Korea. 10th Symposium on Diseases in

Asian Aquaculture (DAA10). Bali, Indonesia, 2017.

2. Cheng Chi, Sib Sankar Giri, Jin Woo Jun, Sang Wha Kim, Jeong Woo Kang,

Hyoun Joong Kim, Saekil Yun, Se Chang Park*. Deep sequencing-based

transcriptome profiling analysis of scallop exposed to marine toxin. 10th

Symposium on Diseases in Asian Aquaculture (DAA10). Bali, Indonesia,

2017.

3. Sib Sankar Giri, Jin Woo Jun, Saekil Yun, Chi Cheng, Hyoun Joong Kim,

Sang Guen Kim, Jeong Woo Kang, Sang Wha Kim, V. Sukumaran*, Se

Chang Park*. Effect of autochthonous bacteria with potential probiotic

properties on physiological conditions of Cyprinus carpio against waterborne

lead toxicity. 10th Symposium on diseases in Asian aquaculture (DAA10).

Bali, Indonesia, 2017.

4. Cheng Chi, Sib Sankar Giri, Jin Woo Jun, Hyoun Joong Kim, Saekil Yun,

Sang Wha Kim, Sang Guen Kim, Se Chang Park*. Effects of Diarrhetic

Shellfish Poisoning Toxins on the Immunological Responses of Bay Scallop

Argopecten irradians. International Conference on Marine Science and

Aquaculture (ICOMSA) 2017. Kota Kinabalu, Malaysia, 2017.

5. Sib Sankar Giri, Jin Woo Jun, Cheng Chi, Hyoun Joong Kim, Sang Guen Kim,

Saekil Yun, Sang Wha Kim, Se Chang Park*. Efficacy of Biosurfactant

Isolated from Bacillus licheniformis VS16 in Mediating Immune Responses in

Major Carp Rohu and its Application in Food Industry. International

64

Conference on Marine Science and Aquaculture (ICOMSA) 2017. Kota

Kinabalu, Malaysia, 2017.

6. Hyoun Joong Kim, Jin Woo Jun, Sib Sankar Giri, Cheng Chi, Saekil Yun,

Sang Guen Kim, Sang Wha Kim, Se Chang Park*. Prophylactic Efficacy of

Bacteriophage to Control Vibrio corallilyticus Infection in Oyster Larvae.

International Conference on Marine Science and Aquaculture (ICOMSA)

2017. Kota Kinabalu, Malaysia, 2017.

7. Jin Woo Jun, Sib Sankar Giri, Cheng Chi, Hyoun Joong Kim, Saekil Yun,

Sang Guen Kim, Sang Wha Kim, Se Chang Park*. Bacteriophage Therapy

for Acute Hepatopancreatic Necrosis Disease (AHPND) in Shrimp.

International Conference on Marine Science and Aquaculture (ICOMSA)

2017. Kota Kinabalu, Malaysia, 2017.

8. Saekil Yun, Sib Sankar Giri, Jin Woo Jun, Hyoun Joong Kim, Cheng Chi,

Sang Guen Kim, Sang Wha Kim, Se Chang Park*. PLGA Microspheres

Loaded with Formalin-killed Aeromonas hydrophila as a Single-shot Vaccine

Against A. hydrophila Infection. International Conference on Marine Science

and Aquaculture (ICOMSA) 2017. Kota Kinabalu, Malaysia, 2017.

9. Sang Guen Kim, Jin Woo Jun, Sib Sankar Giri, Cheng Chi, Hyoun Joong Kim,

Saekil Yun, Sang Wha Kim, Ji Hyung Kim, Se Chang Park*. Isolation and

Characterization of Vibrio Bacteriophage with Anti-biofilm Activity.

International Conference on Marine Science and Aquaculture (ICOMSA)

2017. Kota Kinabalu, Malaysia, 2017.

2016

1. Hyoun Joong Kim, Jin Woo Jun, Sib Sankar Giri, Cheng Chi, Saekil Yun,

Sang Guen Kim, Sang Wha Kim, Se Chang Park*, 中井 敏博*. Application

65

of bacteriophage for controlling Vibrio corallilyticus infection in oyster

hatchery. ファージ・環境ウイルス研究会 合同シンポジウム

プログラム. Yokosuka, Japan, 2016.

2. Jin Woo Jun, Sib Sankar Giri, Cheng Chi, Hyoun Joong Kim, Saekil Yun,

Sang Guen Kim, Sang Wha Kim, Se Chang Park*, 中井 敏博*. Phage

application to aquaculture: phage therapy against shrimp bacterial infectious

disease. ファージ・環境ウイルス研究会 合同シンポジウム

プログラム. Yokosuka, Japan, 2016.

3. Sang Guen Kim, Jin Woo Jun, Sib Sankar Giri, Cheng Chi, Hyoun Joong Kim,

Saekil Yun, Sang Wha Kim, Se Chang Park*, 中井 敏博*. Isolation and

Characterization of Lytic Bacteriophage specific for Vibrio alginolyticus.

ファージ・環境ウイルス研究会 合同シンポジウム プログラム.

Yokosuka, Japan, 2016.

66

Acknowledgements

My postgraduate years were the happiest and most self-fulfilling times of my

entire life. Professor Se Chang Park gave me almost complete freedom of study

and created an environment where I could experiment safely and pleasantly. I'm

very grateful for his help in creating the best situation for me to grow into a

scholar of my own color and to help me develop my skills from the ground up. If

it were not for him, I would not be here today.

What I did during my master's was not something I could do alone. With the

help of so many people, I was able to start and finish this research. More than

anyone else, I offer special thanks to the lab colleagues. Dr. Sib Sankar Giri, Prof.

Jin Woo Jun, Saekil Yun, Hyoun Joong Kim, Sang Guen Kim, Prof. Cheng Chi,

Jeong Woo Kang, Jun Kwon, Se Jin Han, Woo Taek Oh, Dr. Won Hee Hong, and

Dr. Young Ran Lee are all very grateful. It was a great time to learn from each

wise and clever person.

I would like to express my special thanks to Professor Byung Yeop Kim, who

kindly extended a helping hand to me three years ago when I wanted to study

sharks but didn't even know how to get one carcass. With his help, I was able to

continue to challenge myself without giving up my dream. Thank you, always.

I was able to develop my dream as a scholar thanks to my family's sincere

67

support. The reason why I have been able to pursue my dream since I was a young

child is by far the constant support of my mother and father. I know the hard

works that two of you have been done for me to sustain metaphysical

considerations without tripping over the hurdles of reality, and I will never forget

it for the rest of my life. The understanding and respect shown by my sister, Sang

In, and my brother, Sang Won, also helped me greatly in hard times and is one of

the great reasons I could be today. I would like to express my deep gratitude to all

of my family.

I also thank Professor Byung Chun Lee and Dr. Ji Hyung Kim, the committee

for this thesis. Based on the detailed comments and advice, I was able to organize

the final steps of my master's degree successfully.

In addition, I would like to express my sincere thanks to the countless people

who helped me with my research. I will repay everybody’s kindness by

contributing to academia with profound research.

This work was supported by the Ministry of Education, through the Brain

Korea 21 Program for Veterinary Science, Seoul National University and the

Research Institute for Veterinary Science, Seoul National University.